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Labeling of antibiotics for infection diagnosisThe high impact of infection on daily clinical prac-tice has promoted researUniversity Hospital of Bellvitge-IDIBELLoday, 67 MEDICAch into better and more accu-rate diagnostic and therapeutic methods. Localizinginflammation/infection with nuclear medicine tech-niques began over 40 years ago. T AGa-scintig-raphy, 99mTc-nanocolloid, 111In and 99mTc in vitrolabeled leukocytes, and monoclonal antigranulocyteantibodies are widely available for this purpose. While
The early and accurate localization of infectious
foci is a major challenge in contemporary nuclear
these methods are useful for localizing inflammation,
medicine. Early and accurate diagnosis and localiza-
they cannot always differentiate septic from aseptic
tion allow prompt and successful treatment and
processes. The ideal properties of an agent for diag-
decrease associated morbidity. Radiopharmaceuticals
nosing infection include: high specificity, early diag-nosis, rapid blood clearance, ease of preparation, low
such as 67Ga-citrate, in vivo and in vitro labeled leuko-
toxicity, biodistribution appropriate for the disease
cytes, and labeled human immunoglobulins are sen-
under study, absence of immunologic response and
sitive for the diagnosis of inflammation. They are able
low cost. A novel approach to infection diagnosis is the
to detect the physiological and biochemical changes
use of radiolabelled antibiotics. Antibiotics localize in
that occur during the early phases of inflammation.the infectious focus, where they are frequently taken
However, none are capable of reliably differentiating
up and metabolized by microorganisms. The majori-
sterile inflammation from septic infection. Nor are
ty of the various antibiotics studied so far are those of
they are able to identify the presence of the microor-
the quinolones group (ciprofloxacin, sparfloxacin,enrofloxacin, levofloxacin, norfloxacin and ofloxacin).
The ideal properties of an agent for diagnosing
More recently, the labeling of ceftizoxime, a semi-synthetic third generation cephalosporin, has been
infection include: high specificity, early diagnosis,
reported. The relevant features of labeled antibiotics
rapid blood clearance, ease of preparation, low tox-
in research and/or clinical infection diagnosis are the
icity, biodistribution appropriate to the disease under
COPYRIGHTfocus of this article.
study, absence of immunologic response and low
KEY WORDS: Infection - Radionuclide imaging -
cost. Labeling with 99mTc is highly desirable, but oth-
Technetium Tc 99m Sestamibi - Fluoroquinolones -
Ciprofloxacin - Sparfloxacin - Enrofloxacin -
The labeling of antibiotics was introduced about a
decade ago by Solanki et al. in their search for a bet-ter agent to diagnose infection.3 Theoretically, labeledantibiotics would be incorporated and metabolized bythe bacteria present in the infectious focus and, assum-
Address reprint requests to: Benitez A, S. Medicina Nuclear, Hospital
ing that the uptake is proportional to the number of
Universitari de Bellvitge-IDIBELL, 08907 Hospitalet de Llobregat, Spain.E-mail: jmartincomin@csub.scs.es
microorganisms present, the measured radioactivity
THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING
LABELING OF ANTIBIOTICS FOR INFECTION DIAGNOSIS
Figure 1.—Chemical structure of ciprofloxacin.
would accurately and specifically localize the infection.
Figure 2.—Chemical structure of ceftizoxime.
It is important to note that, as always in nuclear med-icine diagnostic procedures, the amount of antibiot-ic used is minimal (tracer doses) and that the new
50.2% and 43.9%, respectively. The uptake of 99mTc-
methyl-diphosphonate used as a control was <2.5%.Similarly, cultures with dead bacteria showed a 99mTc-
maceutical (the labeled antibiotic) does not
arious antibiotics have been studied for this purpose,
most of which are members of the quinolones class
After developing a new formulation, the same
(ciprofloxacin, sparfloxacin, enrofloxacin, levofloxacin,
research group described another labeling method
norfloxacin and ofloxacin). Our group has recently
using stannous ion as the reducing agent.6 The new
introduced the labeling of ceftizoxime, a semisynthet-
formulation was prepared in a kit formulation in 2
ic third generation cephalosporin. The relevant fea-
vials: the one containing the antibiotic solution (2 mg
tures of labeled antibiotics in research and/or clinical
ciprofloxacin) and the other containing the lyophilized
infection diagnosis are the focus of this article.
reducing agent (500 mg stannous tartrate). The label-ing procedure was performed in 2 steps. In the first,400 MBq of freshly eluted 99mTc-pertechnectate were
Radiolabeling antibiotics with 99mTc
added to the vial containing the reducing agent, andthe antibiotic solution was added in the second step.
The first antibiotic to be labeled with 99mTc for infec-
After incubation for 15 min the radiopharmaceutical
tious foci localization was ciprofloxacin, a member
is ready for administration. The high labeling effi-
of the fluoroquinolones group (Figure 1). The label-
ciency (>96%) obviates the purification step.7 This
ing of this antibiotic used formamidine sulphinic acid
new method shortens preparation time and obviates
(FSA) in N2 atmosphere as a 99mTc-reductor agent and
the heating step. Later studies showed that the final
for the union of 99mTc-reduced-cyprofloxacin heating
radiopharmaceutical had in vitro and in vivo proper-
at 100º C for 10 min.3, 5 However, because the FSA was
unstable and, as used in the formulation (2 mg
Following these initial experiences, other fluoro-
ciprofloxacin + 400 mg FSA + 400 MBq 99mTc-pertech-
quinolones have also been labeled with 99mTc:
COPYRIGHT
nectate), the labeling efficiency was inadequate
Levofloxacin,9 Sparfloxacin 10 and Enrofloxacin.11 All
(55±8%), the authors performed a purification step
the procedures use stannous tartrate as the reducing
through a Sephadex DAE 81 column to retain non-
agent, and a kit formulation is envisaged.
reduced anionic free 99mTc. With this purification step,
Another antibiotic that has been radiolabelled is
the labeling efficiency increased to 95% and the com-
ceftizoxime, a third generation semisynthetic cepha-
losporin stable against beta-lactamase (Figure 2).
The bacterial uptake of the labeled antibiotic in
Labeling is performed using sodium dithionite as the
cultures containing Staphylococcus aureus, Pseudo-
reducing agent in mildly alkaline pH (7.8-8). The
monas aeruginosa and Escherichia coli was 58.5%,
99mTc-ceftizoxime complex formation needs heating at
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LABELING OF ANTIBIOTICS FOR INFECTION DIAGNOSIS
100 °C for 100 min. Under these conditions, labeling
Although the labeled antibiotic is not taken up by
efficiency is 94.9±2.4% and in vitro stability is 6 h.
neutrophils or macrophages present in the infectious
Evaluated in agar diffusion plates containing E. coli,
focus, in vitro studies have demonstrated that while
the biological activity of this new compound was 83%
activated neutrophils and macrophages take up 99mTc-
ciprofloxacin, the intracellular concentration dimin-ishes with time.19
99mTc-ciprofloxacin scintigraphy may be more accu-
Analysis of the main 99mTc-labelled antibiotics
rate than labeled leukocytes scintigraphy in the local-
studied for the diagnosis of infection
ization of bacterial infection.5, 13 It approaches theproperties of an ideal tracer; does not require blood
handling, is available in kit form, can be labeled with99mTc, is easy and simple to prepare, and shelf stabil-
99mTc-Ciprofloxacin, a second generation broad spec-
ity is fairly long (8 h).6 As its efficacy does not depend
trum quinolone, was introduced by Solanki et al. in
on the presence of leukocytes, it can be used in
1993. Theirs was the first report of use of a labeled
leukopenic patients. Unlike labeled leukocytes, it is not
antibiotic for diagnosing rather than treating infection.3
taken up to any significant degree by the bone mar-
Venjamuri et al. reported that use of the agent increased
row; it seems to be more accurate than labeled leuko-
the diagnostic specificity of infection and could differ-
cyte scintigraphy in localizing spinal infections.20
entiate sterile from septic inflammation.13
The accumulation of 99mTc-ciprofloxacin in the infec-
tious focus does not appear to be influenced by pre-
vious antibiotic treatment. As published experience is
limited, larger studies on patients with or without pri-
Ciprofloxacin is active against Gram positive and
or antibiotic treatment are necessary to confirm this
Gram negative bacteria. It penetrates the cell through
the membrane channels; once inside the cell, it binds
In a multicenter trial on 500 patients with acute or
to DNA gyrase (topoisomerase II), inhibiting its action.14
chronic inflammation, infection and fever, the diag-
This mechanism applies to Gram negative bacteria,
nosis of infection with the use of 99mTc-ciprofloxacin
whereas in Gram positive bacteria the agent’s action is
had a sensitivity 93%, a specificity of 86%, an accura-
mediated by topoisomerase IV complexes.15, 16 Labeled
cy of 90%, and the positive and negative predictive val-
ciprofloxacin is also active against ciprofloxacin-resis-
Scintigraphic images show a diffuse uptake in the
infectious focus. The uptake can be seen as early as 1 h
postadministration, but later images are a helpful aid to
In the kidneys, bladder, liver and spleen, the early
outline the lesion more accurately. It is interesting that in
activity 99mTc-Ciprofloxacin diminishes in late images.
certain processes such as abscesses labeled leukocytes
Occasionally, gallbladder activity may also be seen
accumulate in the center of the lesion (where most bac-
accompanied by bowel activity. No uptake is seen in
teria are dead), while 99mTc-ciprofloxacin accumulates
brain, normal bone, bone marrow or soft tissue.13, 18
in the periphery (where most bacteria are alive andactive). In other clinical settings such as inflammatorybowel disease, the labeled ciprofloxacin study is usual-
ly negative.17 Results are difficult to standardize. In the cit-
COPYRIGHT
The published results are controversial. Sonmezoglu
ed work, of the 7 patients with chronic inflammation
et al. have applied 99mTc-ciprofloxacin scintigraphy
the scan was negative in 4 (3 with inflammatory bowel
to bone and orthopedic infection with a high accura-
disease and 1 with rheumatoid arthritis), and showed
labeled antibiotic uptake in the remaining 3 (2 with
Initial experiences, both in vitro and in vivo, showed
inflammatory bowel disease and 1 with rheumatoid
that 99mTc-cyprofloxacin localizes at high concentra-
arthritis). Was this related to disease activity at examina-
tions in Gram negative and Gram positive induced
tion? Was there leukocyte antibiotic uptake? While there
abscesses but it does not localize in sterile inflamma-
are no a clear answers to these questions, it is important
to note that the 4-h images normalized at 24 h.
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LABELING OF ANTIBIOTICS FOR INFECTION DIAGNOSIS
According to Larikka et al.,21 24 h imaging reduces
ciprofloxacin by the presence of an amino group in the
the false positive rate and increases specificity from
C-5 position. This amino group seems to be respon-
68% to 95% in patients with total hip prosthesis. This
sible for Sparfloxacin’s greater antimicrobial power.27
early accumulation is due in part to the small size of
It is excreted through liver and kidneys; maximal liv-
the ciprofloxacin molecule that diffuses passively
er and kidney activities at 1, 4 and 24 h postinjection
because of locally increased vascular permeability.
are: 5.7%, 6% and 4.6%, and 7%, 7.1% and 2.2%,
Other false positive results have been described in
patients with fibrous dysplasia, avascular necrosis,6and inflammatory arthroplasties.17
Sonmezoglu et al., in a study using 99mTc-
ciprofloxacin scanning of a group of patients with
Animal and in vitro studies with 99mTc-Sparfloxacin
bone infection, reported 94% sensitivity, 83% specificity
have shown preserved antimicrobial activity against S.
and 89% accuracy. In the authors’ opinion, the rela-
aureus.10 Following the administration of 70 MBq of99m
tive low specificity could have been the result of the
Tc-Sparfloxacin in normal rats, the maximal activ-
ity seen in the liver and kidney decreases with time.
Other authors have reported completely different
The lower hepatobiliary excretion of Sparfloxacin
findings. Sarda et al.7 found that the labeled antibiot-
compared with ciprofloxacin (2.5% versus 6%) may be
ic was unable to differentiate osteomyelitis and sep-
an important aid in the localization of abdominalinfectious foci such as appendicitis.
In inflamed/infected animals, both inflammatory
and infectious foci show uptake in early images (2 h
tic arthritis from other non-septic inflammations.Similarly, a low specificity was found in an S. aureus
prosthesis infection animal model using rabbits.7, 22, 23In a report published in 2002,24 Britton Afound low postinjection), whereas on the later images (4 h) only
sensitivity (60%) in a group of patients with septic
and degenerative arthropathies; the authors suggest-
The higher power of sparfloxacin versus
ed the need to continue research into new more accu-
ciprofloxacin provides a higher uptake of the labeled
antibiotic in the infectious focus, for which higher
In a study on fever of unknown origin 25 the authors
reported high specificity (100%) but low sensitivity
A study to analyze the differences between
(67%). Artiko et al.,26 in their study of abdominal and
sparfloxacin and ciprofloxacin is being conducted on
gastrointestinal infection, also found low sensitivity
patients with chronic osteomyelitis.
These controversial results could be due to differ-
ences in the labeling procedure at the different cen-
Enrofloxacin, another antibiotic of the same
ters using the same antibiotic or to differences in study
Quinolone family with a chemical structure similar
populations or to many other reasons. The results
to ciprofloxacin, is active against Gram positive and
from a multicenter trial now running in the United
States and Canada will help to standardize the agent’s
In a very elegant study, Siaens et al.11 labeled this
antibiotic with 99mTc and compared its efficacy withthat of 99mTc-ciprofloxacin in a group of rats with
intramuscular inflammation or infection: their results
COPYRIGHT
floxacin, a member of a new generation of
showed that none of the labeled quinolones were
quinolones, is more powerful than ciprofloxacin. Like
able to differentiate between sterile inflammation and
its predecessor, Sparfloxacin is active against Gram
infection. In the authors’ opinion, the labeled antibi-
positive and Gram negative bacteria.
otic did not show any specific binding to bacteriaprobably because of changes in the antibiotic structureduring the labeling process.
The scintigrams showed a moderate activity in the
The action of Sparfloxacin is also mediated by its
infectious foci at 1 h postinjection; however, the activ-
inhibitory action on DNA-gyrase but it differs from
ity decreased progressively in the images at 4 h. This
THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING
LABELING OF ANTIBIOTICS FOR INFECTION DIAGNOSIS
nonspecific localization in the infectious focus prob-
mation/infection of different origin have shown 100%
ably results from inflammatory changes associated
sensitivity, 83% specificity and 94%accuracy.32
with infection such as local increase of permeability.In this study, there were no significant differences inthe target/background ratio between inflammation
Conclusions
and infection (S. aureus/Candida albicans). The onlydifference versus ciprofloxacin was the finding of
99mTc-labelled antibiotics have opened a new, excit-
higher liver, spleen and liver activity, which could
ing field of research in infection diagnosis. Published
have been related to the higher lipophilicity of the
results, though controversial, point to the possible
utility of these novel agents in localizing infectious
Other quinolones such as Levofloxacin 9 and
foci and the ability to distinguish such foci from ster-
Norfloxacin 29 have also been labeled with 99mTc, but
experience with these agents is still very limited.
Compared with agents under research or those
recently approved, which are essentially neutrophillabeling procedures, labeled antibiotics represent a
Cephalosporins
potentially significant advance in the diagnosis ofinfection. Because of their biodistribution and excre-
tory pathways, the utility of these agents for diag-
nosing abdominal infection is probably somewhat
Ceftizoxime, a new third generation cephalosporin
limited. Most likely, labeled antibiotics will find the
with beta-lactamase activity, binds to the bacterial
widest application in bone and orthopedic infections.
peptide, inducing an inhibition in cellular wall build-up and eventually bacterial death.30 It is active against
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